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Detection method and detection primer group for rapidly distinguishing mating types of cordyceps militaris

A detection method and technology for detection primers, applied in the biological field, can solve problems such as quality degradation, low efficiency, cumbersome steps, etc., and achieve highly characteristic effects

Pending Publication Date: 2020-12-18
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the strains used in production, it is necessary to clarify the mating types of strains from different sources to avoid unclear characteristics such as some genetic backgrounds. For example, strains that are not single or mixed have two mating types at the same time, resulting in the formation of deformed fruiting bodies in the cultivation of Cordyceps militaris. Serious problems such as quality decline or even production reduction
So far, in order to clarify the mating type of bacteria from different sources, PCR reactions are required to determine the mating type of the strains. This method of identifying the mating type of different strains has low efficiency, and it takes a long time for identification, and the steps are cumbersome. PCR is required. Steps such as pre-deformation, temperature cycling, gel electrophoresis and UV development

Method used

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  • Detection method and detection primer group for rapidly distinguishing mating types of cordyceps militaris
  • Detection method and detection primer group for rapidly distinguishing mating types of cordyceps militaris
  • Detection method and detection primer group for rapidly distinguishing mating types of cordyceps militaris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The primer set Cm1F / Cm1FIP / Cm1BIP / Cm1B detects Cordyceps militaris mating type MAT1-1 strain.

[0022] (1) Quickly release the DNA in the sample;

[0023] Weigh the known mating type as MAT1-1 Cordyceps militaris strain (mating type MAT1-1 represents the strain cm1) fresh sample ≧ 100mg or dry sample ≧ 50mg, put in a mortar, add 3 times the weight of sterile quartz sand, mix After grinding, transfer to a centrifuge tube, add 400-600 μl sterile double-distilled water, bathe in 100°C water for 10 minutes, centrifuge at 10,000 rpm / min for 5 minutes, take the supernatant, and set aside. The supernatant was stored at -20°C and used within 48 hours.

[0024] (2) Loop-mediated isothermal amplification was performed using 4 specific primer sets (Cm1F / Cm1FIP / Cm1BIP / Cm1B) synthesized by Beijing Qingke Biotechnology Co., Ltd. Hunan Branch. The primer sequences are shown in Table 1.

[0025] Table 1 The sequence of primers for rapid detection of Cordyceps militaris mating type MA...

Embodiment 2

[0029] The primer set Cm2F / Cm2FIP / Cm2BIP / Cm2B detects Cordyceps militaris mating type MAT1-2 strain.

[0030] (1) Quickly release the DNA in the sample;

[0031] Weigh the known mating type as the MAT1-2 Cordyceps militaris strain (the mating type MAT1-2 represents the strain cm2) fresh sample ≧ 100mg or dry sample ≧ 50mg, put it in a mortar, add 3 times the weight of sterile quartz sand, mix After grinding, transfer to a centrifuge tube, add 400-600 μl sterile double-distilled water, bathe in 100°C water for 10 minutes, centrifuge at 10,000 rpm / min for 5 minutes, take the supernatant, and set aside. The supernatant was stored at -20°C and used within 48 hours.

[0032] (2) Loop-mediated isothermal amplification was performed using four specific primers (Cm2F / Cm2FIP / Cm2BIP / Cm2B) synthesized by Beijing Qingke Biotechnology Co., Ltd. Hunan Branch. The primer sequences are shown in Table 2.

[0033] Table 2 The sequence of primers for rapid detection of Cordyceps militaris mati...

Embodiment 3

[0038] Different from step (1) in Example 1, the rest are the same. Specifically:

[0039] (1) The DNA of the desired test sample is extracted by CTAB / NaCl method, and the extracted DNA is quantitatively analyzed by 1% agarose gel electrophoresis. Store at -20°C, valid within 1 year.

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Abstract

The invention discloses a method and a detection primer group for rapidly and efficiently detecting mating types of different strains of cordyceps militaris. Specific primers (Cm1F / Cm1FIP / Cm1BIP / Cm1Band Cm2F / Cm2FIP / Cm2BIP / Cm2B) for detecting the mating types MAT1-1 and MAT1-2 of different strains of cordyceps militaris are designed on the basis of an isothermal amplification technology; and the four primers Cm1F / Cm1FIP / Cm1BIP / Cm1B are used for detecting the mating type MAT1-1 of cordyceps militaris, and the other four primers Cm2F / Cm2FIP / Cm2BIP / Cm2B are used for detecting the mating type MAT1-2. The detection result is high in characteristic property, simple and rapid, and the mating types can be rapidly distinguished through the color of the mixed solution only by mixing the reagents andthen placing the reagents in a constant-temperature water bath kettle for reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method for rapidly distinguishing the mating type of Cordyceps militaris and a detection primer set. Background technique [0002] Cordyceps militaris (Cordyceps militaris) is a model species of fungi of the genus Cordyceps, also known as Cordyceps militaris, etc. Hypocreales), Cordycipitaceae (Cordycipitaceae), Cordyceps (Cordyceps). In recent decades, great progress has been made in the research and development of Cordyceps militaris resources, and large-scale artificial cultivation has been realized at present. [0003] Cordyceps militaris is a bipolar heterothallic fungus with two mating types, MAT1-1 and MAT1-2, and its sexual reproduction requires the participation of two mating types. As the strains used in production, it is necessary to clarify the mating types of strains from different sources to avoid unclear characteristics such as some genetic ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 邹娟李婷陈红汛何英杰罗丽媛李嘉嘉刘胜贵
Owner HUAIHUA UNIV
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